Method of determining a chemotherapeutic regimen based on glutathione-s transferase pi expression

ABSTRACT

The present invention relates to prognostic methods which are useful in medicine, particularly cancer chemotherapy. The object of the invention to provide a method for assessing GST-pi expression levels in fixed or fixed and paraffin embedded tissues and prognosticate the probable resistance or sensitivity of a patient&#39;s tumor to treatment with platinum-based therapies by examination of the amount of GST-pi mRNA in a patient&#39;s tumor cells and comparing it to a predetermined threshold expression level. More specifically, the invention provides to oligonucleotide primer pair GST-piand methods comprising their use for detecting levels of GST-pi mRNA.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional application of patent application Ser.No. 09/879,986 filed Jun. 14, 2001, now U.S. Pat. No. 6,686,155, whichis incorporated herein in its entirety by reference thereto.

FIELD OF THE INVENTION

The present invention relates to prognostic methods which are useful inmedicine, particularly cancer chemotherapy. More particularly, theinvention relates to assessment of tumor cell gene expression in apatient. The resistance of tumor cells to chemotherapeutic agents thattarget DNA, especially agents that damage DNA in the manner ofplatinating agents is assayed by examining the mRNA expressed from genesinvolved in DNA repair in humans.

BACKGROUND OF THE INVENTION

Cancer arises when a normal cell undergoes neoplastic transformation andbecomes a malignant cell. Transformed (malignant) cells escape normalphysiologic controls specifying cell phenotype and restraining cellproliferation. Transformed cells in an individual's body thusproliferate, forming a tumor. When a tumor is found, the clinicalobjective is to destroy malignant cells selectively while mitigating anyharm caused to normal cells in the individual undergoing treatment.

Chemotherapy is based on the use of drugs that are selectively toxic(cytotoxic) to cancer cells. Several general classes of chemotherapeuticdrugs have been developed, including drugs that interfere with nucleicacid synthesis, protein synthesis, and other vital metabolic processes.These generally are referred to as antimetabolite drugs. Other classesof chemotherapeutic drugs inflict damage on cellular DNA. Drugs of theseclasses generally are referred to as genotoxic. Susceptibility of anindividual neoplasm to a desired chemotherapeutic drug or combination ofdrugs often, however, can be accurately assessed only after a trialperiod of treatment. The time invested in an unsuccessful trial periodposes a significant risk in the clinical management of aggressivemalignancies.

The repair of damage to cellular DNA is an important biological processcarried out by a cell's enzymatic DNA repair machinery. Unrepairedlesions in a cell's genome can impede DNA replication, impair thereplication fidelity of newly synthesized DNA and/or hinder theexpression of genes needed for cell survival. Thus, genotoxic drugsgenerally are considered more toxic to actively dividing cells thatengage in DNA synthesis than to quiescent, nondividing cells. Normalcells of many body tissues are quiescent and commit infrequently tore-enter the cell cycle and divide. Greater time between rounds of celldivision generally is afforded for the repair of DNA damage in normalcells inflicted by chemotherapeutic genotoxins. As a result, someselectivity is achieved for the killing of cancer cells. Many treatmentregimens reflect attempts to improve selectivity for cancer cells bycoadministering chemotherapeutic drugs belonging to two or more of thesegeneral classes.

Because effective chemotherapy in solid tumors usually requires acombination of agents, the identification and quantification ofdeterminants of resistance or sensitivity to each single drug has becomean important tool to design individual combination chemotherapy.

Two widely used genotoxic anticancer drugs that have been shown todamage cellular DNA are cisplatin (DDP) and carboplatin. Cisplatinand/or carboplatin currently are used in the treatment of selected,diverse neoplasms of epithelial and mesenchymal origin, includingcarcinomas and sarcomas of the respiratory, gastrointestinal andreproductive tracts, of the central nervous system, and of squamousorigin in the head and neck. Cisplatin in combination with other agentsis currently preferred for the management of testicular carcinoma, andin many instances produces a lasting remission. (Loehrer et al.,1984,100Ann. Int. Med. 704). Cisplatin (DDP) disrupts DNA structure throughformation of intrastrand adducts. Resistance to platinum agents such asDDP has been attributed to enhanced tolerance to platinum adducts,decreased drug accumulation, or enhanced DNA repair. Although resistanceto DDP is multifactoral, alterations in DNA repair mechanisms probablyplay a significant role.

The glutathione-S-transferase (GST) family of proteins is involved indetoxification of cytotoxic drugs. By catalyzing the conjugation oftoxic and carcinogenic electrophilic molecules with glutathione the GSTenzymes protect cellular macromolecules from damage (Boyer et al.,Preparation, characterization and properties of glutathioneS-transferases. In: Zakim D, Vessey D (eds.) Biochemical Pharmacologyand Toxicology. New York, N.Y.: John Wiley and Sons, 1985.). A certainisomeric type of these proteins, the glutathione S-transferase Pi(GST-pi, also to be interchangeably refered to as GSTP1 or GST-π herein)is widely expressed in human epithelial tissues and has beendemonstrated to be over-expressed in several tumors (Terrier et al., AmJ Pathol 1990; 137: 845–853; Moscow et al., Cancer Res 1989; 49:1422–1428). Increased GST-pi levels have been found in drug resistanttumors, although the exact mechanism remains unclear (Tsuchida et al.,Crit Rev Biochem Mol Biol 1992; 27: 337–384). Previous studies havesuggested that low expression of GST protein (not mRNA) is associatedwith response to platinum-based chemotherapy (Nishimura et al., Cancer.Clin Cancer Res 1996; 2:1859–1865; Tominaga, et al., Am. J. Gastro.94:1664–1668, 1999; Kase, et al., Acta Cytologia. 42: 1397–1402, 1998).However, these studies did not measure quantitative gene expression, butused a semi-quantitative immunohistochemical staining method to measureprotein levels. However, quantitative GST-pi gene expressionmeasurements are needed to achieve a very effective prognostication.

Most pathological samples are routinely fixed and paraffin-embedded(FPE) to allow for histological analysis and subsequent archivalstorage. Thus, most biopsy tissue samples are not useful for analysis ofgene expression because such studies require a high integrity of RNA sothat an accurate measure of gene expression can be made. Currently, geneexpression levels can be only qualitatively monitored in such fixed andembedded samples by using immunohistochemical staining to monitorprotein expression levels.

Until now, quantitative gene expression studies including those ofGST-pi expression have been limited to reverse transcriptase polymerasechain reaction (RT-PCR) amplification of RNA from fresh or frozentissue.

The use of frozen tissue by health care professionals poses substantialinconveniences. Rapid biopsy delivery to avoid tissue and subsequentmRNA degradation is the primary concern when planning any RNA-basedquantitative genetic marker assay. The health care professionalperforming the biopsy, must hastily deliver the tissue sample to afacility equipped to perform an RNA extraction protocol immediately upontissue sample receipt. If no such facility is available, the clinicianmust promptly freeze the sample in order to prevent mRNA degradation. Inorder for the diagnostic facility to perform a useful RNA extractionprotocol prior to tissue and RNA degradation, the tissue sample mustremain frozen until it reaches the diagnostic facility, however far awaythat may be. Maintenance of frozen tissue integrity during transportusing specialized couriers equipped with liquid nitrogen and dry ice,comes only at a great expense.

Routine biopsies generally comprise a heterogenous mix of stromal andtumorous tissue. Unlike with fresh or frozen tissue, FPE biopsy tissuesamples are readily microdissected and separated into stromal and tumortissue and therefore, offer an advantage over the use of fresh or frozentissue. However, isolation of RNA from fixed tissue, and especiallyfixed and paraffin embedded tissue, results in highly degraded RNA,which is generally not applicable to gene expression studies.

A number of techniques exist for the purification of RNA from biologicalsamples, but none is reliable for isolation of RNA from FPE samples. Forexample, Chomczynski (U.S. Pat. No. 5,346,994) describes a method forpurifying RNA from tissues based on a liquid phase separation usingphenol and guanidine isothiocyanate. A biological sample is homogenizedin an aqueous solution of phenol and guanidine isothiocyanate and thehomogenate thereafter mixed with chloroform. Following centrifugation,the homogenate separates into an organic phase, an interphase and anaqueous phase. Proteins are sequestered in the organic phase, DNA in theinterphase, and RNA in the aqueous phase. RNA can be precipitated fromthe aqueous phase. Unfortunately, this method is not applicable to fixedand paraffin-embedded (FPE) tissue samples.

Other known techniques for isolating RNA typically utilize eitherguanidine salts or phenol extraction, as described for example inSambrook, J. et al., (1989) at pp. 7.3–7.24, and in Ausubel, F. M. etal., (1994) at pp. 4.0.3–4.4.7. Again, none of the known methodsprovides reproducible quantitative results in the isolation of RNA fromparaffin-embedded tissue samples.

Techniques for the isolation of RNA from paraffin-embedded tissues arethus particularly needed for the study of gene expression in tumortissues, since expression levels of certain receptors or enzymes can beused to determine the likelihood of success of a particular treatment.

There is a need for a method of quantifying GST-pi mRNA fromparaffinized tissue in order to provide an early prognosis for proposedgenotoxic cancer therapies. As a result, there has been a concerted yetunsuccessful effort in the art to obtain a quantification of GST-piexpression in fixed and paraffinized (FPE) tissue. Accordingly, it isthe object of the invention to provide a method for assessing GST-pilevels in tissues fixed and paraffin-embedded (FPE) and prognosticatethe probable resistance of a patient's tumor to treatment with DNAdamaging agents, creating the type of lesions in DNA that are created byDNA platinating agents, by examination of the amount of GST-pi mRNA in apatient's tumor cells and comparing it to a predetermined thresholdexpression level.

SUMMARY OF THE INVENTION

In one aspect of the invention there is provided a method for assessinglevels of expression of GST-pi mRNA obtained from fixed andparaffin-embedded (FPE) fixed and paraffin-embedded (FPE) tumor cells.

In another aspect of the invention there is provided a method ofquantifying the amount of GST-pi mRNA expression relative to an internalcontrol from a fixed and paraffin-embedded (FPE) tissue sample. Thismethod includes isolation of total mRNA from said sample and determiningthe quantity of GST-pi mRNA relative to the quantity of an internalcontrol gene's mRNA.

In an embodiment of this aspect of the invention, there are providedoligonucleotide primers having the sequence of GST-F (SEQ ID NO: 1) orGST-R (SEQ ID NO:2) and sequences substantially identical thereto. Theinvention also provides for oligonucleotide primers having a sequencethat hybridizes to SEQ ID NO: 1 or SEQ ID NO:2 or their complementsunder stringent conditions.

In yet another aspect of the invention there is provided a method fordetermining a chemotherapeutic regimen for a patient, comprisingisolating RNA from a fixed and paraffin-embedded (FPE) tumor sample;determining a gene expression level of GST-pi in the sample; comparingthe GST-pi gene expression levels in the sample with a predeterminedthreshold level for the GST-pi gene; and determining a chemotherapeuticregimen based on results of the comparison of the GST-pi gene expressionlevel with the predetermined threshold level.

The invention further relates to a method of normalizing the uncorrectedgene expression (UGE) of GST-pi relative to an internal control gene ina tissue sample analyzed using TaqMan® technology to known GST-piexpression levels relative to an internal control from samples analyzedby pre-TaqMan® technology.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows and association between survival and GST-pi correctedrelative mRNA expression in patients with esophagocardiac adenocarcinomatreated with 5-FU and cisplatin. Patients with GST-pi values above themedian/threshold value had a survival advantage compared to those withpatients with values below the median/threshold. Censored values aredenoted by a tick.

FIG. 2 is a graph showing survival analysis confined to patients withTNM Stage II esophagocardiac adenocarcinoma

FIG. 3 is a graph showing survival analysis confined to patients withStage IV esophagocardiac adenocarcinoma

FIG. 4 is a chart illustrating how to calculate GST-pi expressionrelative to an internal control gene. The chart contains data obtainedwith two test samples, (unknowns 1 and 2), and illustrates how todetermine the uncorrected gene expression data (UGE). The chart alsoillustrates how to normalize UGE generated by the TaqMan® instrumentwith known relative GST-pi values determined by pre-TaqMan® technology.This is accomplished by multiplying UGE to a correction factorK_(GST-pi). The internal control gene in the figure is β-actin and thecalibrator RNA is Human Liver Total RNA (Stratagene, Cat. #735017).

FIG. 5 shows the oligonucleotide primers used in the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention resides in part in the finding that the amount ofGST-pi mRNA is correlated with increased sensitivity to DNA platinatingagents. Tumors expressing high levels of GST-pi mRNA are consideredlikely to be sensitive to platinum-based chemotherapy. Conversely, thosetumors expressing low amounts of GST-pi mRNA are likely to beinsensitive to platinum-based chemotherapy. A patient's relativeexpression of tumor GST-pi mRNA is judged by comparing it to apredetermined threshold expression level. Such sensitivity or lackthereof to DNA platinating agents is determined by a patient'ssurvivability.

The invention relates to a method of quantifying the amount of GST-pimRNA expression in fixed and paraffin-embedded (FPE) tissue relative togene expression of an internal control. The present inventors havedeveloped oligonucleotide primers that allow accurate assessment ofGST-pi expression in tissues that have been fixed and embedded. Theinvention oligonucleotide primers, GST-F (SEQ ID NO: 1), GST-R (SEQ IDNO: 2), or oligonucleotide primers substantially identical thereto,preferably are used together with RNA extracted from fixed and paraffinembedded (FPE) tumor samples. This measurement of GST-pi gene expressionmay then be used for prognosis of platinum-based chemotherapy.

This embodiment of the invention involves first, a method for reliableextraction of RNA from an FPE sample and second, determination of thecontent of GST-pi mRNA in the sample by using a pair of oligonucleotideprimers, preferably oligionucleotide primer pair GST-F (SEQ ID NO: 1)and GST-R (SEQ ID NO: 2), or oligonucleotides substantially identicalthereto, for carrying out reverse transcriptase polymerase chainreaction. RNA is extracted from the FPE cells by any of the methods formRNA isolation from such samples as described in U.S. patent applicationSer. No. 09/469,338, filed Dec. 20, 1999, now U.S. Pat. No. 6,248,535,and is hereby incorporated by reference in its entirety.

The present method can be applied to any type of tissue from a patient.For examination of sensitivity of tumor tissue, it is preferable toexamine the tumor tissue. In a preferred embodiment, a portion of normaltissue from the patient from which the tumor is obtained, is alsoexamined.

The methods of the present invention can be applied over a wide range oftumor types. This allows for the preparation of individual “tumorexpression profiles” whereby expression levels of GST-pi are determinedin individual patient samples and response to various chemotherapeuticsis predicted. Preferably, the methods of the invention are applied tosolid tumors, most preferably esophogocardiac tumors. For application ofsome embodiments of the invention to particular tumor types, it ispreferable to confirm the relationship of GST-pi gene expression levelsto clinical resistance by compiling a data-set that enables correlationof a particular GST-pi expression and clinical resistance toplatinum-based chemotherapy.

A “predetermined threshold level”, as defined herein, is a level ofGST-pi expression above which it has been found that tumors are likelyto be sensitive to a platinum-based chemotherapeutic regimen. Expressionlevels below this threshold level are likely to be found in tumorsinsensitive to platinum-based chemotherapeutic regimen. The range ofcorrected relative expression of GST-pi, expressed as a ratio of GST-pi:β-actin, among tumors responding to a platinum-based chemotherapeuticregimen is more than about 1.0×10⁻³. Tumors that do not respond to aplatinum-based chemotherapeutic regimen have relative expression ofGST-pi: β-actin ratio below about 1.0×10⁻³. FIG. 1. However, the presentinvention is not limited to the use of β-actin as an internal controlgene.

In performing the method of this embodiment of the present invention,tumor cells are preferably isolated from the patient. Solid or lymphoidtumors or portions thereof are surgically resected from the patient orobtained by routine biopsy. RNA isolated from frozen or fresh samples isextracted from the cells by any of the methods typical in the art, forexample, Sambrook, Fischer and Maniatis, Molecular Cloning, a laboratorymanual, (2nd ed.), Cold Spring Harbor Laboratory Press, New York,(1989). Preferably, care is taken to avoid degradation of the RNA duringthe extraction process.

However, tissue obtained from the patient after biopsy is often fixed,usually by formalin (formaldehyde) or gluteraldehyde, for example, or byalcohol immersion. Fixed biological samples are often dehydrated andembedded in paraffin or other solid supports known to those of skill inthe art. Non-embedded, fixed tissue may also be used in the presentmethods. Such solid supports are envisioned to be removable with organicsolvents for example, allowing for subsequent rehydration of preservedtissue.

RNA is extracted from the FPE cells by any of the methods as describedin U.S. patent application Ser. No. 09/469,338, filed Dec. 20, 1999, nowU.S. Pat. No. 6,248,535, which is hereby incorporated by reference inits entirety. Fixed and paraffin-embedded (FPE) tissue samples asdescribed herein refers to storable or archival tissue samples. RNA maybe isolated from an archival pathological sample or biopsy sample whichis first deparaffinized. An exemplary deparaffinization method involveswashing the paraffinized sample with an organic solvent, such as xylene,for example. Deparaffinized samples can be rehydrated with an aqueoussolution of a lower alcohol. Suitable lower alcohols, for exampleinclude, methanol, ethanol, propanols, and butanols. Deparaffinizedsamples may be rehydrated with successive washes with lower alcoholicsolutions of decreasing concentration, for example. Alternatively, thesample is simultaneously deparaffinized and rehydrated. RNA is thenextracted from the sample.

For RNA extraction, the fixed or fixed and deparaffinized samples can behomogenized using mechanical, sonic or other means of homogenization.Rehydrated samples may be homogenized in a solution comprising achaotropic agent, such as guanidinium thiocyanate (also sold asguanidinium isothiocyanate). Homogenized samples are heated to atemperature in the range of about 50 to about 100° C. in a chaotropicsolution, which contains an effective amount of a chaotropic agent, suchas a guanidinium compound. A preferred chaotropic agent is guanidiniumthiocyanate.

An “effective concentration of chaotropic agent” is chosen such that atan RNA is purified from a paraffin-embedded sample in an amount ofgreater than about 10-fold that isolated in the absence of a chaotropicagent. Chaotropic agents include: guanidinium compounds, urea,formamide, potassium iodiode, potassium thiocyantate and similarcompounds. The preferred chaotropic agent for the methods of theinvention is a guanidinium compound, such as guanidinium isothiocyanate(also sold as guanidinium thiocyanate) and guanidinium hydrochloride.Many anionic counterions are useful, and one of skill in the art canprepare many guanidinium salts with such appropriate anions. Theeffective concentration of guanidinium solution used in the inventiongenerally has a concentration in the range of about 1 to about 5 M witha preferred value of about 4 M. If RNA is already in solution, theguanidinium solution may be of higher concentration such that the finalconcentration achieved in the sample is in the range of about 1 to about5 M. The guanidinium solution also is preferably buffered to a pH ofabout 3 to about 6, more preferably about 4, with a suitable biochemicalbuffer such as Tris-Cl. The chaotropic solution may also containreducing agents, such as dithiothreitol (DTT) and β-mercaptoethanol(BME). The chaotropic solution may also contain RNAse inhibitors.

Homogenized samples may be heated to a temperature in the range of about50 to about 100° C. in a chaotropic solution, which contains aneffective amount of a chaotropic agent, such as a guanidinium compound.A preferred chaotropic agent is guanidinium thiocyanate.

RNA is then recovered from the solution by, for example, phenolchloroform extraction, ion exchange chromatography or size-exclusionchromatography. RNA may then be further purified using the techniques ofextraction, electrophoresis, chromatography, precipitation or othersuitable techniques.

The quantification of GST-pi mRNA from purified total mRNA from fresh,frozen or fixed is preferably carried out using reverse-transcriptasepolymerase chain reaction (RT-PCR) methods common in the art, forexample. Other methods of quantifying of GST-pi mRNA include forexample, the use of molecular beacons and other labeled probes useful inmultiplex PCR. Additionally, the present invention envisages thequantification of GST-pi mRNA via use of PCR-free systems employing, forexample fluorescent labeled probes similar to those of the Invader®Assay (Third Wave Technologies, Inc.). Most preferably, quantificationof GST-pi cDNA and an internal control or house keeping gene (e.g.β-actin) is done using a fluorescence based real-time detection method(ABI PRISM 7700 or 7900 Sequence Detection System [TaqMan®], AppliedBiosystems, Foster City, Calif.) or similar system as described by Heidet al., (Genome Res 1996;6:986–994) and Gibson et al.(Genome Res1996;6:995–1001). The output of the ABI 7700 (TaqMan® Instrument) isexpressed in Ct's or “cycle thresholds”. With the TaqMan® system, ahighly expressed gene having a higher number of target molecules in asample generates a signal with fewer PCR cycles (lower Ct) than a geneof lower relative expression with fewer target molecules (higher Ct).

As used herein, a “house keeping” gene or “internal control” is meant toinclude any constitutively or globally expressed gene whose presenceenables an assessment of GST-pi mRNA levels. Such an assessmentcomprises a determination of the overall constitutive level of genetranscription and a control for variations in RNA recovery.“House-keeping” genes or “internal controls” can include, but are notlimited to the cyclophilin gene, β-actin gene, the transferrin receptorgene, GAPDH gene, and the like. Most preferably, the internal controlgene is β-actin gene as described by Eads et al., Cancer Research 1999;59:2302–2306.

A control for variations in RNA recovery requires the use of “calibratorRNA.” The “calibrator RNA” is intended to be any available source ofaccurately pre-quantified control RNA. Preferably, Adult Colon, DiseaseHuman Total RNA, (Cat. No. #735263) from Stratagene, is used.

“Uncorrected Gene Expression (UGE)” as used herein refers to the numericoutput of GST-pi expression relative to an internal control genegenerated by the TaqMan® instrument. The equation used to determine UGEis shown in Example 3, and illustrated with sample calculations in FIG.4.

A further aspect of this invention provides a method to normalizeuncorrected gene expression (UGE) values acquired from the TaqMan®instrument with “known relative gene expression” values derived fromnon-TaqMan® technology. Preferably, the known non-TaqMan® derivedrelative GST-pi: β-actin expression values are normalized with TaqMan®derived GST-pi UGE values from a tissue sample.

“Corrected Relative GST-pi Expression” as used herein refers tonormalized GST-pi expression whereby UGE is multiplied with a GST-pispecific correction factor (K_(GST-pi)), resulting in a value that canbe compared to a known range of GST-pi expression levels relative to aninternal control gene. Example 3 and FIG. 4 illustrate thesecalculations in detail. These numerical values allow the determinationof whether or not the “Corrected Relative GST-pi Expression” of aparticular sample falls above or below the “predetermined threshold”level. The predetermined threshold level of Corrected Relative GST-piExpression to β-actin level is about 1.0×10⁻³. K_(GST-pi) specific forGST-pi, the internal control β-actin and calibrator Adult Colon, DiseaseHuman Total RNA, (Cat. No. #735263) from Stratagene, is 7.28×10⁻³.

“Known relative gene expression” values are derived from previouslyanalyzed tissue samples and are based on the ratio of the RT-PCR signalof a target gene to a constitutively expressed internal control gene(e.g. β-Actin, GAPDH, etc.). Preferably such tissue samples are formalinfixed and paraffin-embedded (FPE) samples and RNA is extracted from themaccording to the protocol described in Example 1 and in U.S. patentapplication Ser. No. 09/469,338, filed Dec. 20, 1999, now U.S. Pat. No.6,248,535, which is hereby incorporated by reference in its entirety. Toquantify gene expression relative to an internal control standardquantitative RT-PCR technology known in the art is used. Pre-TaqMan®technology PCR reactions are run for a fixed number of cycles (i.e., 30)and endpoint values are reported for each sample. These values are thenreported as a ratio of GST-pi expression to β-actin expression. See U.S.Pat. No. 5,705,336 to Reed et al.

K_(GST-pi) may be determined for an internal control gene other thanβ-actin and/or a calibrator RNA different than Adult Colon, DiseaseHuman Total RNA, (Cat. No. #735263) from Stratagene. To do so, one mustcalibrate both the internal control gene and the calibrator RNA totissue samples for which GST-pi expression levels relative to thatparticular internal control gene have already been determined (i.e.,“known relative gene expression”). Preferably such tissue samples areformalin fixed and paraffin-embedded (FPE) samples and RNA is extractedfrom them according to the protocol described in Example 1 and in U.S.patent application Ser. No. 09/469,338, filed Dec. 20, 1999, now U.S.Pat. No. 6,248,535, which is hereby incorporated by reference in itsentirety. Such a determination can be made using standard pre-TaqMan®,quantitative RT-PCR techniques well known in the art. Upon such adetermination, such samples have “known relative gene expression” levelsof GST-pi useful in the determining a new K_(GST-pi) specific for thenew internal control and/or calibrator RNA as described in Example 3.

The methods of the invention are applicable to a wide range of tissueand tumor types and so can be used for assessment of clinical treatmentof a patient and as a diagnostic or prognostic tool for a range ofcancers including breast, head and neck, lung, esophageal, colorectal,and others. In a preferred embodiment, the present methods are appliedto prognosis of esophagocardiac adenocarcinoma.

Pre-chemotherapy treatment tumor biopsies are usually available only asfixed paraffin embedded (FPE) tissues, generally containing only a verysmall amount of heterogeneous tissue. Such FPE samples are readilyamenable to microdissection, so that GST-pi gene expression may bedetermined in tumor tissue uncontaminated with stromal tissue.Additionally, comparisons can be made between stromal and tumor tissuewithin a biopsy tissue sample, since such samples often contain bothtypes of tissues.

Generally, any oligonucleotide pair that flanks a region of GST-pi genemay be used to carry out the methods of the invention. Primershybridizing under stringent conditions to a region of the GST-pi genefor use in the present invention will amplify a product between 20–1000base pairs, preferably 50–100 base pairs, most preferably less than 100base pairs.

The invention provides specific oligonucleotide primers pairs andoligonucleotide primers substantially identical thereto, that allowparticularly accurate assessment of GST-pi expression in FPE tissues.Preferable are oligonucleotide primers, GST-F (SEQ ID NO: 1) and GST-R(SEQ ID NO: 2), (also referred to herein as the oligonucleotide primerpair GST) and oligonucleotide primers substantially identical thereto.The oliogonucleotide primers GST-F (SEQ ID NO: 1) and GST-R, (SEQ ID NO:2) have been shown to be particularly effective for measuring GST-pimRNA levels using RNA extracted from the FPE cells by any of the methodsfor mRNA isolation, for example as described Example 1 and in U.S.patent application Ser. No. 09/469,338, filed Dec. 20, 1999, now U.S.Pat. No. 6,248,535, which is hereby incorporated by reference in itsentirety.

“Substantially identical” in the nucleic acid context as used herein,means hybridization to a target under stringent conditions, and alsothat the nucleic acid segments, or their complementary strands, whencompared, are the same when properly aligned, with the appropriatenucleotide insertions and deletions, in at least about 60% of thenucleotides, typically, at least about 70%, more typically, at leastabout 80%, usually, at least about 90%, and more usually, at least,about 95–98% of the nucleotides. Selective hybridization exists when thehybridization is more selective than total lack of specificity. See,Kanehisa, Nucleic Acids Res., 12:203–213 (1984).

This invention includes substantially identical oligonucleotides thathybridize under stringent conditions (as defined herein) to all or aportion of the oligonucleotide primer sequence of GST-F (SEQ ID NO: 1),its complement or GST-R (SEQ ID NO: 2), or its complement.

Under stringent hybridization conditions, only highly complementary,i.e., substantially similar nucleic acid sequences hybridize.Preferably, such conditions prevent hybridization of nucleic acidshaving 4 or more mismatches out of 20 contiguous nucleotides, morepreferably 2 or more mismatches out of 20 contiguous nucleotides, mostpreferably one or more mismatch out of 20 contiguous nucleotides.

The hybridizing portion of the nucleic acids is typically at least 10(e.g., 15) nucleotides in length. The hybridizing portion of thehybridizing nucleic acid is at least about 80%, preferably at leastabout 95%, or most preferably about at least 98%, identical to thesequence of a portion or all of oligonucleotide primer GST-F (SEQ ID NO:1), its complement or GST-R (SEQ ID NO: 2), or its complement.

Hybridization of the oligonucleotide primer to a nucleic acid sampleunder stringent conditions is defined below. Nucleic acid duplex orhybrid stability is expressed as a melting temperature (T_(m)), which isthe temperature at which the probe dissociates from the target DNA. Thismelting temperature is used to define the required stringencyconditions. If sequences are to be identified that are substantiallyidentical to the probe, rather than identical, then it is useful tofirst establish the lowest temperature at which only homologoushybridization occurs with a particular concentration of salt (e.g. SSCor SSPE). Then assuming that 1% mismatching results in a 1° C. decreasein T_(m), the temperature of the final wash in the hybridizationreaction is reduced accordingly (for example, if sequences having >95%identity with the probe are sought, the final wash temperature isdecrease by 5° C.). In practice, the change in T_(m) can be between 0.5°C. and 1.5° C. per 1% mismatch.

Stringent conditions involve hybridizing at 68° C. in 5×SSC/5×Denhart'ssolution/1.0% SDS, and washing in 0.2×SSC/0.1% SDS at room temperature.Moderately stringent conditions include washing in 3×SSC at 42° C. Theparameters of salt concentration and temperature be varied to achieveoptimal level of identity between the primer and the target nucleicacid. Additional guidance regarding such conditions is readily availablein the art, for example, Sambrook, Fischer and Maniatis, MolecularCloning, a laboratory manual, (2nd ed.), Cold Spring Harbor LaboratoryPress, New York, (1989) and F. M. Ausubel et al eds., Current Protocolsin Molecular Biology, John Wiley and Sons (1994).

Oligonucleotide primers disclosed herein are capable of allowingaccurate assessment of GST-pi gene expression in a fixed or fixed andparaffin embedded tissue, as well as frozen or fresh tissue. This isdespite the fact that RNA derived from FPE samples is more fragmentedrelative to that of fresh or frozen tissue. Thus, the methods of theinvention are suitable for use in assaying GST-pi expression levels inFPE tissue where previously there existed no way to assay GST-pi geneexpression using fixed tissues.

From the measurement of the amount of GST-pi mRNA that is expressed inthe tumor, the skilled practitioner can make a prognosis concerningclinical resistance of a tumor to a particular genotoxin, preferably aplatinum-based chemotherapy, or to a chemotherapy inducing a similartype of DNA damage. Platinum-based chemotherapies cause a “bulky adduct”of the DNA, wherein the primary effect is to distort thethree-dimensional conformation of the double helix. Such compounds aremeant to be administered alone, or together with other chemotherapiessuch as gemcitabine (Gem) or 5-Fluorouracil (5-FU).

Many compounds are commonly given with platinum-based chemotherapyagents. For example, BEP (bleomycin, etoposide, cisplatin) is used fortesticular cancer, MVAC (methotrexate, vinblastine, doxorubicin,cisplatin) is used for bladder cancer, MVP (mitomycin C, vinblastine,cisplatin) is used for non-small cell lung cancer treatment. Manystudies have documented interactions between platinum-containing agents.Therapeutic drug synergism, for example, has been reported for manydrugs potentially included in a platinum based chemotherapy. A veryshort list of recent references for this include the following: Okamotoet al., Urology 2001; 57:188–192.; Tanaka et al., Anticancer Research2001; 21:313–315; Slamon et al., Seminars in Oncology 2001; 28:13–19;Lidor et al., Journal of Clinical Investigation 1993; 92:2440–2447;Leopold et al., NCI Monographs 1987;99–104; Ohta et al., Cancer Letters2001; 162:39–48; van Moorsel et al., British Journal of Cancer 1999;80:981–990.

Platinum-based genotoxic chemotherapies comprises heavy metalcoordination compounds which form covalent DNA adducts. Generally, theseheavy metal compounds bind covalently to DNA to form, in pertinent part,cis-1,2-intrastrand dinucleotide adducts. Generally, this class isrepresented by cis-diamminedichloroplatinum (II) (cisplatin), andincludes cis-diammine-(1,1-cyclobutanedicarboxylato) platinum(II)(carboplatin), cis-diammino-(1,2-cyclohexyl) dichloroplatinum(II), andcis-(1,2-ethylenediammine) dichloroplatinum(II). Platinum first agentsinclude analogs or derivatives of any of the foregoing representativecompounds.

Tumors currently manageable by platinum coordination compounds includetesticular, endometrial, cervical, gastric, squamous cell,adrenocortical and small cell lung carcinomas along withmedulloblastomas and neuroblastomas. Trans-Diamminedichloroplatinum (II)(trans-DDP) is clinically useless owing, it is thought, to the rapidrepair of its DNA adducts. The use of trans-DDP as a chemotherapeuticagent herein likely would provide a compound with low toxicity innonselected cells, and high relative toxicity in selected cells. In apreferred embodiment, the platinum compound is cisplatin.

The invention being thus described, practice of the invention isillustrated by the experimental examples provided below. The skilledpractitioner will realize that the materials and methods used in theillustrative examples can be modified in various ways. Suchmodifications are considered to fall within the scope of the presentinvention.

EXAMPLES Example 1 RNA Isolation from FPE Tissue

RNA is extracted from paraffin-embedded tissue by the following generalprocedure.

A. Deparaffinization and Hydration of Sections:

(1) A portion of an approximately 10 μM section is placed in a 1.5 mLplastic centrifuge tube.

(2) 600 μL, of xylene are added and the mixture is shaken vigorously forabout 10 minutes at room temperature (roughly 20 to 25° C.).

(3) The sample is centrifuged for about 7 minutes at room temperature atthe maximum speed of the bench top centrifuge (about 10–20,000×g).

(4) Steps 2 and 3 are repeated until the majority of paraffin has beendissolved. Two or more times are normally required depending on theamount of paraffin included in the original sample portion.

(5) The xylene solution is removed by vigorously shaking with a loweralcohol, preferably with 100% ethanol (about 600 μL) for about 3minutes.

(6) The tube is centrifuged for about 7 minutes as in step (3). Thesupernatant is decanted and discarded. The pellet becomes white.

(7) Steps 5 and 6 are repeated with successively more dilute ethanolsolutions: first with about 95% ethanol, then with about 80% and finallywith about 70% ethanol.

(8) The sample is centrifuged for 7 minutes at room temperature as instep (3). The supernatant is discarded and the pellet is allowed to dryat room temperature for about 5 minutes.

B. RNA Isolation with Phenol-Chloroform

(1) 400 μL guanidine isothiocyanate solution including 0.5% sarcosineand 8 μL dithiothreitol is added.

(2) The sample is then homogenized with a tissue homogenizer(Ultra-Turrax, IKA-Works, Inc., Wilmington, N.C.) for about 2 to 3minutes while gradually increasing the speed from low speed (speed 1) tohigh speed (speed 5).

(3) The sample is then heated at about 95° C. for about 5–20 minutes. Itis preferable to pierce the cap of the tube containing the sample with afine gauge needle before heating to 95° C. Alternatively, the cap may beaffixed with a plastic clamp or with laboratory film.

(4) The sample is then extracted with 50 μL 2 M sodium acetate at pH 4.0and 600 μL of phenol/chloroform/isoamyl alcohol (10:1.93:0.036),prepared fresh by mixing 18 mL phenol with 3.6 mL of a 1:49 isoamylalcohol:chloroform solution. The solution is shaken vigorously for about10 seconds then cooled on ice for about 15 minutes.

(5) The solution is centrifuged for about 7 minutes at maximum speed.The upper (aqueous) phase is transferred to a new tube.

(6) The RNA is precipitated with about 10 μL glycogen and with 400 μLisopropanol for 30 minutes at −20° C.

(7) The RNA is pelleted by centrifugation for about 7 minutes in abenchtop centrifuge at maximum speed; the supernatant is decanted anddiscarded; and the pellet washed with approximately 500 μL of about 70to 75% ethanol.

(8) The sample is centrifuged again for 7 minutes at maximum speed. Thesupernatant is decanted and the pellet air dried. The pellet is thendissolved in an appropriate buffer for further experiments (e.g., 50 pI.5 mM Tris chloride, pH 8.0).

Example 2

mRNA Reverse Transcription and PCR

Reverse Transcription: RNA was isolated from microdissected ornon-microdissected formalin fixed paraffin embedded (FPE) tissue asillustrated in Example 1 and as previously described in U.S. applicationSer. No. 09/469,338 filed Dec. 20, 1999, now U.S. Pat. No. 6,248,535,which is hereby incorporated by reference in its entirety. Afterprecipitation with ethanol and centrifugation, the RNA pellet wasdissolved in 50 μl of 5 mM Tris/Cl at pH 8.0. M-MLV ReverseTranscriptase will extend an oligonucleotide primer hybridized to asingle-stranded RNA or DNA template in the presence of deoxynucleotides,producing a complementary strand. The resulting RNA was reversetranscribed with random hexamers and M-MLV Reverse Transcriptase fromLife Technologies. The reverse transcription was accomplished by mixing25 μl of the RNA solution with 25.5 μl of “reverse transcription mix”(see below). The reaction was placed in a thermocycler for 8 min at 26°C. (for binding the random hexamers to RNA), 45 min at 42° C. (for theM-MLV reverse transcription enzymatic reaction) and 5 min at 95° C. (forheat inactivation of DNAse).

“Reverse transcription mix” consists of 10 ul 5×buffer (250 mM Tris-HCl,pH 8.3, 375 mM KCl, 15 mM MgCl2), 0.5 ul random hexamers (50 O.D.dissolved in 550 ul of 10 mM Tris-HCl pH 7.5) 5 ul 10 mM dNTPs (dATP,dGTP, dCTP and dTTP), 5 ul 0.1 M DTT, 1.25 ul BSA (3 mg/ml in 10 mMTris-HCL, pH 7.5), 1.25 ul RNA Guard 24,800 U/ml (RNAse inhibitor)(Porcine #27-0816, Amersham Pharmacia) and 2.5 ul MMLV 200 U/ul (LifeTech Cat #28025-02).

Final concentrations of reaction components are: 50 mM Tris-HCl, pH 8.3,75 mM KCl, 3 mM MgCl2, 1.0 mM dNTP, 1.0 mM DTT, 0.00375. mg/ml BSA, 0.62U/ul RNA Guard and 10 U/ul MMLV.

PCR Quantification of mRNA expression. Quantification of GST-pi cDNA andan internal control or house keeping gene (e.g., β-actin) cDNA was doneusing a fluorescence based real-time detection method (ABI PRISM 7700 or7900 Sequence Detection System [TaqMan®], Applied Biosystems, FosterCity, Calif.) as described by Heid et al., (Genome Res 1996;6:986–994);Gibson et al., (Genome Res 1996;6:995–1001). In brief, this method usesa dual labelled fluorogenic TaqMan® oligonucleotide probe, (GST-219T(SEQ ID NO: 3), T_(m)=69° C.), that anneals specifically within theforward and reverse primers. Laser stimulation within the capped wellscontaining the reaction mixture causes emission of a 3′ quencher dye(TAMRA) until the probe is cleaved by the 5′ to 3′ nuclease activity ofthe DNA polymerase during PCR extension, causing release of a 5′reporter dye (6FAM). Production of an amplicon thus causes emission of afluorescent signal that is detected by the TaqMan®'s CCD (charge-coupleddevice) detection camera, and the amount of signal produced at athreshold cycle within the purely exponential phase of the PCR reactionreflects the starting copy number of the sequence of interest.Comparison of the starting copy number of the sequence of interest withthe starting copy number of the internal control gene provides arelative gene expression level. TaqMan® analyses yield values that areexpressed as ratios between two absolute measurements (gene ofinterest/internal control gene).

The PCR reaction mixture consisted 0.5 μl of the reverse transcriptionreaction containing the cDNA prepared as described above 600 nM of eacholigonucleotide primer (GST-F (SEQ ID NO:1), T_(m)=59° C. and GST-R (SEQID NO: 2), T_(m)=59° C.), 200 nM TaqMan® probe (SEQ ID NO:3), 5 UAmpliTaq Gold Polymerase, 200 μM each DATP, dCTP, dGTP, 400 μM dTTP, 5.5mM MgCl₂, and 1×TaqMan® Buffer A containing a reference dye, to a finalvolume of less than or equal to 25 μl (all reagents Applied Biosystems,Foster City, Calif). Cycling conditions were, 95° C. for 10 min,followed by 45 cycles at 95° C. for 15 s and 60° C. for 1 min.Oligonucleotides used to quantify internal control gene β-Actin wereβ-Actin TaqMan® probe (SEQ ID NO: 4), β-Actin-592F (SEQ ID NO: 5) andβ-Actin-651R (SEQ ID NO: 6).

The oligonucleotide primers GST-F (SEQ ID NO:1) and GST-R (SEQ ID NO:2), used in the above described reaction will amplify a 72 bp product.

Example 3

Determining the Uncorrected Gene Expression (UGE) for GST-pi

Two pairs of parallel reactions are carried out, i.e., “test” reactionsand the “calibration” reactions. The GST-pi amplification reaction andthe β-actin internal control amplification reaction are the testreactions. Separate GST-pi and β-actin amplification reactions areperformed on the calibrator RNA template and are referred to as thecalibration reactions. The TaqMan® instrument will yield four differentcycle threshold (Ct) values: Ct_(GST-pi) and Ct_(β-actin) from the testreactions and Ct_(GST-pi) and Ct_(β-actin) from the calibrationreactions. The differences in Ct values for the two reactions aredetermined according to the following equation:ΔCt _(test) =Ct _(GST-pi) −Ct _(β-actin)(From the “test” reaction)ΔCt _(calibrator) =Ct _(GST-pi) −Ct _(βactin)(From the “calibration”reaction)

Next the step involves raising the number 2 to the negative ΔCt,according to the following equations.2^(−ΔCt) _(test)(From the “test” reaction)2^(−ΔCt) _(calibrator)(From the “calibration” reaction)

In order to then obtain an uncorrected gene expression for GST-pi fromthe TaqMan® instrument the following calculation is carried out:Uncorrected gene expression (UGE) for GST-pi=2^(−ΔCt) _(test)/2^(−ΔCt)_(calibrator)

Normalizing UGE with Known Relative GST-pi Expression Levels

The normalization calculation entails a multiplication of the UGE with acorrection factor (K_(GST-pi)) specific to GST-pi and a particularcalibrator RNA. A correction factor K_(GST-pi) can also be determinedfor any internal control gene and any accurately pre-quantifiedcalibrator RNA. Preferably, the internal control gene β-actin and theaccurately pre-quantified calibrator Adult Colon, Disease Human TotalRNA, (Cat. No. #735263) from Stratagene, are used. Given these reagentscorrection factor K_(GST-pi) equals 7.28×10⁻³.

Normalization is accomplished using a modification of the ΔCt methoddescribed by Applied Biosystems, the TaqMan® manufacturer, in UserBulletin #2 and described above. To carry out this procedure, the UGE of6 different test tissues was analyzed for GST-pi expression using theTaqMan® methodology described above. The internal control gene β-actinand the calibrator RNA,Adult Colon, Disease Human Total RNA, (Cat. No.#735263) from Stratagene was used.

The known relative GST-pi expression level of each sample 14-1, 14-5,14-8, 13-24, 13-25 was divided by its corresponding TaqMan® derived UGEto yield an unaveraged correction factor K.K _(unaveraged)=Known Values/UGE

Next, all of the K values are averaged to determine a single K_(GST-pi)correction factor specific for GST-pi, Adult Colon, Disease Human TotalRNA, (Cat. No. #735263) from Stratagene from calibrator RNA and β-actin.

Therefore, to determine the Corrected Relative GST-pi Expression in anunknown tissue sample on a scale that is consistent with pre-TaqMan®GST-pi expression studies, one merely multiplies the uncorrected geneexpression data (UGE) derived from the TaqMan® apparatus with theK_(GST-pi) specific correction factor, given the use of the sameinternal control gene and calibrator RNA.Corrected Relative GST-pi Expression=UGE×K _(GST-pi)

A K_(GST-pi) may be determined using any accurately pre-quantifiedcalibrator RNA or internal control gene. Future sources of accuratelypre-quantified RNA can be calibrated to samples with known relativeGST-pi expression levels as described in the method above or may now becalibrated against a previously calibrated calibrator RNA such as AdultColon, Disease Human Total RNA, (Cat. No. #735263) from Stratagenedescribed above.

For example, if a subsequent K_(GST-pi) is determined for a differentinternal control gene and/or a different calibrator RNA, one mustcalibrate both the internal control gene and the calibrator RNA totissue samples for which GST-pi expression levels relative to thatparticular internal control gene have already been determined. Such adetermination can be made using standard pre-TaqMan®, quantitativeRT-PCR techniques well known in the art. The known expression levels forthese samples will be divided by their corresponding UGE levels todetermine a K for that sample. K values are then averaged depending onthe number of known samples to determine a new K_(GST-pi) specific tothe different internal control gene and/or calibrator RNA.

Example 4

GST-pi Expression Correlates with Survivability

Total mRNA was isolated from microdissected FPE pretreatment tumorsamples, and Corrected Relative GST-pi Expression was measured usingquantitative RT-PCR as described in Examples 2 and 3. A method for mRNAisolation from such samples is described in Example 1 and in U.S. patentapplication Ser. No. 09/469,338, filed Dec. 20, 1999, now U.S. Pat. No.6,248,535, and is hereby incorporated by reference in its entirety.

The values of the gene expressions were correlated with clinical outcomeusing appropriate statistical methods. Survival was estimated accordingto Kaplan and Meier (Kaplan et al., J Am Stat Assoc 1958; 53: 187–220).Univariate analysis was performed with the log-rank test (Mantel,Chemother Rep 1966; 50: 163–170). The level of significance was set toP<0.05. All P values reported were based on two-sided tests.

A total of 31 esophageal or gastroesophageal junction (esophagocardiac)adenocarcinoma tumor specimens from 31 patients were analysed for GST-pimRNA expression analysis. Thirty (97%) of the patients were male, themedian age was 64 years (mean 60.9 years, range 36–78 years). The ethnicbackground of this group included 29 Caucasians, 1 Asian, and 1African-American. Using TNM clinical staging criteria, 2 (6.5%) of thepatients had Stage I disease, 22 (71%) had Stage II disease, 1 (3.2%)had Stage III disease, and 6 (19.4%) patients had Stage IV disease.Overall survival was assessable for all patients. The median overallsurvival was 17.17 months (mean 24.8 months, range 3.8–156.7 months).Twelve (38.7%) of the patients had died and 19 (61.3%) were alive.

The treatment consisted of all patients receiving two cycles of 5-FUgiven as 800 mg/m² per day for 5 days or 1000 mg/m² per day for 4 daysplus 75 mg/m² cisplatin with concurrent 45 Gy radiation, followed byoperative resection. For entry into the study, each patient had to havecompleted the chemotherapy regimen and the prescribed radiotherapy,undergone a gross complete resection, and lived at least 30 days aftersurgery.

The influence of tumor stage was accounted for by pooling the data overthe TNM stage strata. Survival curves and log-rank statistics weregenerated for Stage II and Stage IV disease patients only because of thevery small numbers of patients in the other stages. The median correctedrelative GST-pi mRNA expression level was 1.0×10⁻³ (mean 0.51×10⁻³,range 0.0–16.1×10⁻³, all values GST-pi×10⁻³/β-actin). An analysis ofsurvival according to GST-pi values showed that patients whose tumorshad a relative GST-pi gene expression level higher than the median valuehad a statistically significant survival benefit compared to those withlevels below the median value (P=0.0073, log-rank test). Accordingly,the median corrected relative GST-pi mRNA was assigned to be a thresholdvalue. This association is shown graphically in FIG. 1. The relationshipwas independent of stage. FIGS. 2 and 3 show that the association waspresent if the analysis was confined only to those with Stage II orStage IV disease.

GST-pi mRNA expression is a significant prognostic factor for patientswith esophagocardiac adenocarcinoma who are treated with acisplatin-containing regimen.

1. An oligonucleotide primer having the sequence of SEQ ID NO:1, whereinsaid primer is capable of amplifying a portion of GST-pi mRNA isolatedfrom fixed and paraffin embedded (FPE) tumor tissue when used with SEQID NO:
 2. 2. An oligonucleotide primer having the sequence of SEQ IDNO:2, wherein said primer is capable of amplifying a portion of GST-pimRNA isolated from fixed and paraffin embedded (FPE) tumor tissue whenused with SEQ ID NO:
 1. 3. A kit for detecting expression of a GST-pigene comprising the oligonucleotide primers of claim 1 and claim 2.